Distribution of thrombospondins and their neuronal receptor α2δ1 in the rat retina
Introduction
The retina is an easily accessible part of the central nervous system (CNS) and often serves as a model system for the study of brain injury and repair. Various injury conditions, including ischemia-reperfusion induced by elevated intraocular pressure (IOP), retinal detachment, and oygen-induced retinopathy (Chen et al., 2008; Sethi et al., 2005; Fu et al., 2011; Dorfman et al., 2011; Dijk et al., 2007), could induce plastic changes of retinal synapses, which may be the direct cause of post-injury visual function impairment. Abnormal synaptic changes may explain why visual function cannot recover as effectively as neuronal survival following many interventions. Therefore, it is necessary to study the protection of synapses and processes that rescue neurons from death following retinal injury and repair; however, the potential molecular mechanisms triggering synaptic plasticity after retinal injury still remain unclear. Glial cells have been shown to participate in the regulation of synaptic transmission and plasticity (Volterra and Meldolesi, 2005; Mauch et al., 2001; Henneberger et al., 2010). Emerging evidences have demonstrated that thrombospondin (TSP) 1 and TSP2 expressed by immature and reactive glial cells were responsible for excitatory CNS synaptogenesis or synapse formation through an interaction with their neuronal receptor, the calcium channel subunit α2δ1 (Christopherson et al., 2005; Eroglu et al., 2009; Procko and Shaham, 2009).
Thrombospondins (TSPs) are large oligomeric, multidomain, extracellular matrix proteins that have been previously shown to play important roles in cell attachment, cell migration, cytoskeletal dynamics and angiogenesis. TSPs mediate these functions via their interactions with various cell surface receptors through specific domains (Tan and Lawler, 2009; Kyriakides and Maclauchlan, 2009). There are five TSPs which are separated into two subfamilies, A and B, according to their overall organization. TSP1 and TSP2 belong to the subgroup A family and are assembled as trimers. They share the same structural and functional domains. It was recently shown that in the developing CNS, TSP1 and TSP2 promoted the formation of new synapses (Christopherson et al., 2005; Crawford et al., 2012). Alpha-2-delta (α2δ) is a membrane-spanning auxiliary protein subunit of voltage-gated calcium channels (VGCCs) that is found in muscle and brain, where it plays an essential role in controlling neurotransmitter release, neuronal excitability and gene expression (Taylor and Garrido, 2008). The α2δ family consists of at least four distinctive genes, α2δ1, α2δ2, α2δ3 and α2δ4. A key feature of α2δ1 is that it is considered to be the high-affinity receptor for two commonly used anti-epileptic drugs, gabapentin and pregabalin. Recently, α2δ1 and α2δ3 have been shown to have novel functions in synaptogenesis independent of their effects on calcium channels (Kurshan et al., 2009; Bauer et al., 2010).
It has also been widely reported that glial cells in the retina were distinctly activated in response to glaucoma, retinal ischemia-reperfusion injury induced by elevated IOP, and photoreceptor degeneration (Middeldorp and Hol, 2011; Lorber et al., 2012; Grosche et al., 1995). It is unknown whether reactive glial cells are responsible for the synaptic plasticity process that is induced by retinal injury through secreting TSPs that interact with α2δ1. We speculated that TSP1/2 is a likely modulator of synaptic plasticity after retinal injury; however, little is known about the specific cellular localization and distribution pattern of TSP1/2 and their receptor α2δ1 in retinal tissue.
In the present study, we identified in detail the immunohistochemical localization of TSP1/2 and their neuronal receptor α2δ1 in adult rat retinas. We also further investigated the changes in TSP1/2 and α2δ1 expression after injury through a retinal ischemia-reperfusion model induced by elevated IOP.
Section snippets
Animals
Fifteen healthy adult Sprague–Dawley rats (no limit of male or female) weighing 200–250 g were acquired in-house from the animal center of Hunan Agricultural University, China (license No. SCXK (Xiang) 2009-0012). The animals were given tap water and food in an environmentally controlled room at a temperature of 25 °C and a relative humidity of 50%–60% with a 12-h:12-h light–dark cycle (light on from 7:00–19:00). The animal protocol was in compliance with the guidelines of the EU Directive
Immunolocalization of TSP1 in the adult rat retina
In the rat retina, TSP1 was greatly distributed in the GCL, where TSP1 immunoreactivity was expressed only in the membrane and cytoplasm of each cell (Fig. 1A). Mild staining was also observed in the cell bodies of the inner nuclear layer (INL). No TSP1 immunoreactivity, however, was visible in the outer nuclear layer (ONL). The pigment epithelial cells exhibited fairly strong dense labeling.
To identify whether TSP1 expression occurred in glial cells in the retina, double immunofluorescence
Discussion
TSP1 and TSP2 have been termed matrix cellular proteins and implicated in a variety of processes, including cell adhesion, cell migration, and developmental and pathological angiogenesis through interacting with several cell surface receptors and other extracellular matrix proteins (Tan and Lawler, 2009). In addition, TSP1 is a major component of platelet a-granules, while TSP2 is typically a product of fibroblasts (Kyriakides and Maclauchlan, 2009). Most notably, evidence has emerged that both
Acknowledgments
This work was sponsored by the National Natural Science Foundation of China, No. 81070729; the Doctoral Foundation of Ministry of Education of China, No. 20100162110067; and the Natural Science Foundation of Hunan Province, No. 10JJ4023 and supported by the Hunan Provincial Innovation Foundation For Postgraduate, No. CX2011B047.
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